Novel double-stranded ribonucleic acids with rugged physico-chemical structure and highly specific biologic activity

ABSTRACT

A novel form of Rugged dsRNA with a unique composition and physical characteristics was identified with high specificity of binding to TLR3, which conveys an important range of therapeutic opportunities. Unlike the previous known antiviral Ampligen® (poly I, poly C12,U) the new and improved form (poly I, poly C 30 ,U) has a reduced tendency to form branched dsRNA which results in increased bioactivity due to an increased ability to bind TLR3 receptor. Pharmaceutical formulations containing the new nucleic acid as active ingredients and methods of treatment are also provided. The invention also provides a description of the physicochemical properties of this novel form of Rugged dsRNA and a method for its preparation in substantially pure form. DsRNAs acting thru TLR3 receptor activation are potent antiviral compounds as well as anticancer agents; also through secondary immunomodulation they can enhance the bioactivity of vaccines and also treat autoimmune disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. application Ser. No.12/591,270, filed Nov. 13, 2009, which is a continuation-in-part ofInternational Application No. PCT/US2009/005797, filed Oct. 23, 2009which claims priority benefit of U.S. provisional application, Ser. No.61/193,030, filed Oct. 23, 2008 the disclosures of all of which arehereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to our discovery of a novel and improveddouble-stranded ribo-nucleic acid (dsRNA) having specific biologicalactivities, which includes acting as a selective agonist for activationof Toll-like receptor 3 (TLR3). Its smaller and “rugged” molecularstructure as measured by physico-chemical techniques is resistant tomolecular unfolding (i.e., denaturation) and branching. This structureappears to be respon-sible for increased efficacy of dsRNA intherapeutic applications and improved biological activity (e.g., used asan immunoregulatory agent).

BACKGROUND OF THE INVENTION

Ampligen® poly(I):poly(C₁₂U) was developed as a syn-theticdouble-stranded ribonucleic acid (dsRNA) for therapeutic applicationsbased on an understanding of both the beneficial and adverse effectsinduced by poly(I):poly(C) on the physiology of a subject. Acting on thehypothesis that the nucleotide sequence requirements for beneficial andadverse effects are different, poly(I):poly(C₁₂U) was developed by us topreserve the beneficial aspects of dsRNA without the adverse effects ofpoly(I):poly(C) by modifying the latter's structure with the occasionalintroduction of uridylate into the poly(C) strand to produce duplexescontaining specifically-configured regions which are not base paired(i.e., “mismatched”) at the position of the modification. These regionsaccelerate dsRNA hydrolysis and lessen toxicity (Greene, 1984). On theother hand, the ability to induce interferon synthesis was retained aslong as the modified dsRNA persisted for a half life of at least fiveminutes and the frequency of random insertion into thepoly(ribocytidylic acid) strand was not greater than each 0.5 to 1.0helical turn of perfectly base-paired dsRNA (Brodsky, 1987).

While poly(I):poly(C₁₂U) is stable in solution, it is susceptible tohydrolysis like all other conventional nucleic acids. The hydrolysis ishighly dependent on nucleic acid structure, as well as on the presenceof nuclease and divalent cations, pH, and temperature. RNA is moresusceptible to hydrolysis than DNA because of the 2′-OH group present inthe former that facilitates hydrolysis. Moreover, poly(I):poly(C₁₂U) wasdesigned to degrade more rapidly than other dsRNA in anuclease-containing environment, such as blood and other tissue fluids.Nucleic acids are initially stable in physiological salt buffers at roomtemperature, but gradually begin to degrade with time. This hydrolysisrate is temperature dependent, increasing greatly at highertemperatures.

Properties of poly(I):poly(C₁₂U) are characterized by physico-chemicalassays as shown in Table 1. Circular dichroism (CD) (e.g., ellipticity,melting behavior) is used to characterize the double-helical RNAstructure, which is critical to potency. Briefly, Toll-like receptor 3(TLR3) is activated by dsRNA (Alexopoulou, 2001), which leads to a hostdefense recruitment sequence, ultimately producing type I interferons(Schroeder, 2005), initiation of interferon production by dsRNA bindingto TLR3 requires RNA helical structure (Bell, 2006). Although X-raydiffraction and NMR alone are the definitive techniques to determine RNAsecond-order structure, CD measurement with a combination of scanningand thermal stress modes also can provide precise characterization ofthe critical double-helical structure. Indeed, minor changes insecond-order structure of polynucleotides have been measured by CD(Gray, 1995), including the effects of ligand binding (Sumita, 2005).

TABLE 1 Biological Activity and Measured Attributes. Measured PropertyIdentity Attribute Activity Attribute Conformation: Second Degree CD:Ellipticity Double-Stranded RNA: binding to TLR3 integrity of helix CD:Melting Behavior Double Stranded RNA: binding to TLR3 Melting Point ½Width Integrity and uniformity binding to TLR3 of helix Composition andSize Maximum Size No. of Repeat Units Tendency to form BranchedStructure C:U Ratio Identity Tendency to form Branched StructureTherefore, circular dichroism can be employed to characterize thetherapeutic potency of specifically-configured dsRNAs includingpoly(I):poly(C₁₂U) and a now improved dsRNA called Rugged dsRNA.

A problem of Ampligen®, poly(I): poly (C12U), is its lower than expectedbiological activity traced to a branching structure. Our invention isthe unexpected discovery of a new family of improved dsRNAs having aspecific physico-chemical structure and highly specific biologicalactivities, which includes acting as a selective agonist for TLR3. Thisinvention relates to the discovery of this new and improved version ofdsRNA with a superior biological and therapeutic profile. The new andimproved dsRNA, called Rugged dsRNA, can be present in trace amountswithin the Ampligen® mixture. A method is disclosed to enrich the RuggeddsRNA species so it becomes the dominant structure. Its rugged structureas measured by physico-chemical techniques is resistant to molecularunfolding (i.e., denaturation). Improvement in at least one or morebiological activities may result, from the rugged structure of thisparticular form of dsRNA. Other advantages and improvements aredescribed below or would be appar-ent from the disclosure herein.

The Eli Lilly and Company, Patent No. USRE 39,071E is an example of anewly discovered biochemical/biological intermediate in existingunimproved biochemical/biological mixtures of drugs resulting inpatentability. (See also U.S. Pat. No. 6,468,967 and U.S. Pat. No.6,852,689.)

SUMMARY OP THE INVENTION

It is an objective of the invention to provide new and improved forms ofdouble-stranded ribonucleic acid (dsRNA). Their physico-chemicalstructure and biolo-gical activities are described herein. A “rugged”dsRNA molecule resistant to unfolding (i.e., denaturation) of itshelical structure and a reduced tendency to form branched dsRNAmolecular structures and having an improved dsRNA activity as aselective agonist of Toll-like receptor 3 (TLR3). At least partialpurification of Rugged dsRNA from other dsRNA present after synthesis isexpected to increase specificity in its use as a medicament and therebyreduce adverse effects attributable to the dsRNA that is not rugged.

Specifically-configured Ampligen® dsRNA mixture may be of the generalformula ribo(I_(n)).ribo(C₁₁₋₁₄U)_(n), or ribo(I_(n)).ribo(C₁₂U)_(n),wherein the strands are comprised of ribonucleotides (ribo) and n is aninteger from about 500 to about 2,000 repeats. For example, a strandcomprised of poly(ribo-cytosinic₁₁₋₁₄uracilic acid), orpoly(ribocytosinic₁₂uracilic acid) may be partially hybridized to anopposite strand comprised of poly(riboinosinic acid) such that the twostrands form an RNA double helix that is not paired at the uracil base(i.e., mismatch).

After synthesis, Rugged dsRNA may be isolated from the Ampligen® mixtureby at least subjecting the partially hybridized strands of a populationof dsRNA to conditions that denature most dsRNA (at least 50 mol %, atleast 80 mol %, at least 90 mol %, or at least 95 mol %) in thepopulation, and then selection negatively or positively (or both) fordsRNA that remain partially hybridized. The purity of Rugged dsRNA maythus be increased from less than about 1-12 mol % (e.g., less than about12 mol %) relative to all RNA in the population after synthesis. It ispreferred that the Rugged dsRNA be more than about 80-98 mol % relativeto all RNA present in the same mixture with the Rugged dsRNA (at least80 mol %, at least 90 mol %, at least 95 mol %, or at least 98 mol %)after selection. The denaturing conditions to unfold at least partiallyhybridized strands of dsRNA may comprise appropriate choice of buffersalts, pH, solvent, temperature, or any combination thereof. Conditionsmay be empirically determined by observation of the unfolding or meltingof the duplex strands of ribonucleic acid. The yield of Rugged dsRNA maybe improved by partial hydrolysis of longer strands of ribonucleic acid,then selection of (partially) hybridized stands of appropriate size andresistance to denaturation.

The molecular weight of Rugged dsRNA may be from about 30 Kda to about300 Kda, or from about 75 Kda to about 225 Kda. Lengths of a single orboth strands of Rugged dsRNA may be from about 50 bases to about 500bases, or from about 126 bases to about 375 bases. The number of helicalturns made by duplexed RNA strands of Rugged dsRNA may be from about 4.7to about 46.7, or from about 11.7 to about 35 helical turns.

In another aspect, at least one or more different Rugged dsRNA may beadministered to a subject (e.g., human patient or animal) in need ofsuch treatment. Rugged dsRNA may be administered at a dosage of fromabout 0.5 μg to about 600 mg/dose. This dosage may be administered onceper week or month, or two or more doses per week or month. Each dose(e.g., from about 0.5 μg to about 600 mg, from about 1 mg to about 100mg, or from about 10 mg to about 40 mg) may be provided to the subjectwithout limitation to the formulation of the pharmaceutical composition,or its route of administration (although intravenous infusion ispreferred). Use of an effective amount of Rugged dsRNA to achieve afeeling of improved health and may be continued until at least onesymptom is improved. The effective amount required to obtain suchimprove-ment may be identical to or higher than the amount required formaintenance of the effect(s).

The Rugged dsRNA may act specifically through a TLR3 receptor. Thefunction and phenotype of dendritic cells may be normalized in a subject(e.g., human patient or animal). Administering at least an effectiveamount of one or more Rugged dsRNA to a subtest (e.g., human patient oranimal) may thereby decrease the number or reduce the severity ofsymptoms when the subject is afflicted by a disease or otherpathological condition. Use of Rugged dsRNA may correct dendritic cellmaturation abnormalities in the subject without the hazard of inducing acytokine storm.

Antigen presenting cells (e.g., dendritic cells, macrophages, B cells)and mucosal tissues (e.g., gastric or respiratory epithelium) arepreferred targets in the body for Rugged dsRNA. One or more antigens maybe presented to cells of the immune system, and the antigen(s) should besusceptible to the action of the Rugged dsRNA acting selectively as aTLR3 agonist. Cells of the immune system, microbes, cancer cells, orother transformed cells may be susceptible to specific cytokine responsepatterns activated by Rugged dsRNA acting selec-tively as a TLR3agonist. The Rugged dsRNA is preferably administered by intravenousinfusion; intradermal subcutaneous, or intramuscular injection;intranasal or intratracheal inhalation; or oropharyngeal or sublingualapplication.

In another aspect, a medicament is provided as a pharmaceuticalcomposition. One or more different Rugged dsRNA may be used for theirbeneficial effect(s) on a subject's health, as selective TLR3agonist(s), to treat a disease or other pathological condition, or tomanufacture medicaments or pharmaceutical compositions to treat adisease or ether pathological condition. Optional inert ingredients ofthe composition include excipients and a vehicle (e.g., saline buffer orwater) as a single dose or a multi-dose package (e.g., an injection vialor vials), and instructions for their use. Processes for making andusing the pharmaceutical composition (medicament) are also provided. Forexample, one or more different Rugged dsRNA may be formulated at aconcentration from about 0.05 mg/mL to about 0.25 mg/mL (e.g. 10 mgdissolved in 4 mL or 20 mg dissolved in 8 mL) in physiologicalphosphate-buffered saline and stored at from 2° C. to 8° C. in arefrigerator under aseptic conditions.

Further aspects of the invention will be apparent from our descriptionof specific embodiments and the appended claims, and generalizationsthereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an HPLC chromatogram for poly(I):poly(C₁₂U). The minorpeak (not integrated) centered at a retention time of about 5.00 min isRugged dsRNA. The first major peak centered at a retention time of about7.58 min is the single-stranded poly(C₁₂U). The second major peakcentered at a retention time of about 10.05 min is the single-strandedpoly(I). The molecular identity of each peak was determined byphotodiode array (PDA) analysis.

FIG. 1B is an HPLC chromatogram for lyophilized poly(I):poly(C₁₂U)showing aggregates lyophilization can produce. Note that aggregationdoes not occur in the solution process of FIG. 1C, which avoidslyophilization.

FIG. 1C shows a HPLC chromatogram of a sterile solution of poly I: poly(C₁₂U) also showing a novel 5 minute peak.

FIGS. 2A, 2B and 2C show PDA analyses of the three HPLC peaks.Acetonitrile which is used as a solvent, is responsible for the strongabsorbance at 230 nm. Absorbance at 245 nm indicates the presence ofpoly(I); absorbance at 265 nm indicates the presence of poly(C₁₂U). FIG.2A is PDA analysis of the peak centered at a retention time of about5.01 min, which contains both poly(I) and poly(C₁₂U) character. FIG. 2Bis PDA analysis of the peak centered at a retention time of about 7.58min, which contains poly(C₁₂U). FIG. 2C is PDA analysis of the peakcentered at a retention time of about 10.05 min, which contains poly(I).

FIG. 3 is a circular dichroism (CD) of poly(I):poly (C₁₂U). The meltingpoint of 64° C. represents the condition of ½ double stranded structure.

FIG. 4 is the CD wavelength scan of poly(I):poly(C₁₂U). The doublestranded structure is characterized by two peaks at 245 nm and 278 nm,representing two chiral centers normally present in fully doublestranded poly I:poly (C₁₂U). These centers represent chirality due tobase pair structure (278 nm) and the base stacking which is associatedwith the formation of duplex double helix.

FIG. 5 shows the circular dichroism of poly(I):poly(C₁₂U) with thecharacteristic chiral peaks at 245 nm and 278 nm

FIG. 6 shows a plot of the derivative of the thermal melt ofpoly(I):poly (C₁₂U). Integrity of the structure is characterized by themelting point and the ½ width of this derivative profile, both expressedas degrees C.

FIG. 7 shows by HPLC that preparation with heating abolishes all doublestrand structure as reflected by loss of 245 nm peak seen in FIG. 4since the 245 nm peak is due to chiral base stacking. However, analysisby circular dichroism shows that, as a product of thermal stress, the 5minute peak maintains both double helix configuration and chiral centersin the backbone.

FIG. 8 shows a CD plot of a thermal melt of single stranded poly(I). Thechiral center for inosine provides a weak signal at about 252 nm.

FIG. 9 shows a plot of the derivative of the thermal melt of singlestranded poly(I) There is no evidence of intra molecular base stackingat thermal condition which would otherwise disrupt a double helix.

FIG. 10 shows a CD plot of a thermal melt of single stranded poly(C₁₂U). A strong signal is apparent due to the chirality of cytidine.However, the absence of a second peak at 245 nm shows that intramolecular base stacking of Poly C12U does not occur.

FIG. 11 shows a plot of the derivative of the thermal melt of singlestranded poly (C₁₂U). There is no evidence of intra molecular basestacking at thermal condition which would otherwise disrupt a doublehelix.

FIG. 12 shows a CD plot of a thermal melt of poly(I):poly(C₁₂U) andpoly(I):poly(C). Base stacking is evident in both compounds as indicatedby the peak at 245 nm.

FIG. 13 shows a plot of the derivative of the thermal melt ofpoly(I):poly(C₁₂U) and poly(I):poly(C). Both compounds exhibit thecritical melting point for disruption of the double helix. However, thelower melting point of Poly I:Poly C₁₂U illustrates a more labilecharacter which in turn affords the advantageous safety profile of theuridine substituted compound.

FIG. 14 shows a CD wavelength scan of poly(I):poly(C₁₂U) andpoly(A):poly(U). A very weak and shifted single structure may beassociated with the propensity for chiral aggregation of poly(A):poly(U)

FIG. 15 shows a plot of the derivative of the thermal melt ofpoly(I):poly(C₁₂U) and poly(A):poly(U). Somewhat high melting point islikely related to the aggregation tendency of poly(A):poly(U) noted inFIG. 14.

FIG. 16 shows the derivative of a thermal melt of single stranded poly(I):poly(C₁₀U). The greater degree of Uridine substitution (comparepoly(I):poly(C₁₂U, FIG. 6) has compromised the double helical structure.The 1:12 ratio of U:C is optimal, providing one interruption per helicalturn.

FIG. 17 shows a CD plot of thermal melt of single stranded poly(I):poly(C₁₀U). Consistent with the lack of thermal melt behavior (FIG.16), the greater degree of Uridine substitution (1:10 ratio of U:C, cf.1:12 in poly(I):poly(C₁₂U) has abolished the base stacking signal at 245nm.

FIGS. 18A, 18B, 18C show size exclusion chromatography of complexes ofTLR3-ECD and poly(I):poly(C₁₂U) (FIG. 18A), the receptor TLR3-ECD only(FIG. 18B), and the ligand poly(I):poly(C₁₂) only (FIG. 18C).

FIG. 19 shows the effect of thermal stress (40° C.) on the size of dsRNAas measured by analytical centrifugation. The decrease in sedimentationcoefficient (S_(20,w)) reflects a loss of size due to hydrolysis.

FIG. 20 shows the effect of thermal stress (40° C.) upon the componentstrands of dsRNA (7 minute and 10 minute peaks) and the Rugged dsRNA asmeasured by high performance liquid chromatography (HPLC). Whereas thethe larger poly(I) and poly(C₁₂U) strands hydrolyze at 40° C., thequantity of Rugged dsRNA peak increases.

FIG. 21 shows the relative size of Ampligen® vs new Rugged dsRNA (peak 5minutes).

FIG. 22. Partial view of poly(I):poly(C₁₂U) partially hybridized strandsand the interaction of bases of individual poly(I) and the poly (C₁₂U)strands. Molecular weight 1,100,000 da.

FIG. 23. Partial view of poly(I):poly(C₁₂U) partially hybridized strandsand the interaction of bases of individual poly(I) and the poly (C₁₂U)strands. Molecular weight 286,000 da.

FIG. 24. is a lateral view of Rugged dsRNA (a minor component in theunimproved Ampligen® mixture) bound to the active site of the TLR3homodimer (2 horseshoe shaped structures). The C-terminal regions ofeach dimer face each other and bind to the phosphate backbone of thedsRNA. The N-terminals of each TLR3 bind to opposite ends of the dsRNAwith a minimum length of 50 bp required for interaction with essentialresidues of TLR3 for activation of intracellular signaling. Amino acidsof TLR3 required for binding of Rugged dsRNA are shown using Van derWaals' radii associated with the phosphate backbone.

FIG. 25. Illustrates the TLR3 homodimer complexed with Rugged dsRNA (aminor component in the unimproved Ampligen® mixture) as seen down thelong axis of the dsRNA.

FIG. 26. Shows a typical example of a branched dsRNA structure containedin the unimproved Ampligen® mixture.

FIG. 27 shows two typical examples of the branched dsRNA structurecontained in the unimproved Ampligen® mixture.

FIG. 28 shows a typical example of a more completed branched dsRNAstructure contained in the unimproved Ampligen® mixture.

FIG. 29 shows typical examples of unbranched dsRNA molecules containedin the new/improved Rugged dsRNA.

DESCRIPTION OF SPECIFIC EMBODIMENTS Definitions

dsRNA

Doable-stranded (ds) RNA (ribonucleic acid) is chemically very similarto DNA (deoxyribonucleic acid). It is also a long molecule containingnucleotides linked together by 3′-5′ phoshodiestor bonds. Twodifferences in its chemical groups distinguish dsRNA from DNA. The firstis a minor modification of sugar component. The sugar of DNA isdeoxyribose, where as RNA contains ribose, which is identical todeoxyribose except for the presences of an additional hydroxyl group.The second difference is that RNA contains no thymine, but insteadcontains the closely related pyrimidine, uracil. DsRNA terms from thehybridization of two complementary polyribonucleotides forming a doublehelix similar to that of DNA. The two strands of the double helix areheld together by hydrogen-bonded base pairs.

Ampligen®

Ampligen® is a particular dsRNA denoted Poly I: Poly C₁₂U, wherein oneof the two polyribonucleotides is polyriboinosinic acid and the other ispolyribocytidylic₁₂, uridylic acid. Thus, the pyrimidine building blocksof Ampligen® are present in a ratio of 12

cytosines of each uracil, while the complementary purine strand contains13 inosine residues. Within the double-stranded helical structure ofAmpligen® the pyrimidine, cytosine, hydrogen bonds with the purine,inosine, while the pyrimidine, uracil, does not form any hydrogen bonds.Therefore, a “mismatch” is created once for every 12 base pairs (bps)formed between the inosine and cytosine residues. In contrast toAmpligen®, Poly I: Poly C contains only complementary inosine: cytosinebase pairs. No uracil is present in Poly I: Poly C and there are nomismatches.

TLR3 (Toll-Like Receptor 3)

TLR3 is a receptor for a form of immunity called “innate immunity” whichrecognizes double-stranded RNAs with a minimum size of at least 50 basepairs. The size requirement or discrimination of dsRNA by TLB3 preventsresponses to non-microbial sources of dsRNA micro (ml) RNA or transfer(t) RNA. TLR3 exists as a horseshoe shaped monomer with a N-terminalligand-binding extra-cytoplasmic domain (ECD), a transmembrane domain(TMD), and a C-terminal cytoplasmic signaling domain (CSD). X-raycrystallographic studies have provided structural data for the TLR-3ligand complex which consists of a TLR3 homo-dimer complexed to dsRNA ofat least about 50 consecutive base pairs. The formation of the complex(FIG. 24) is believed to transmit a conformational change in the CSD viathe TMD connector that allows cytoplasmic signaling. Above 50 basepairs, binding affinity is a function of size with a progressiveincrease in binding affinity with increased length in linearnon-branched dsRNA.

Rugged dsRNA

Rugged dsRNA is a novel form of dsRNA with a unique composition andphysical characteristics. Unlike the previously known antiviral,Ampligen® (Poly I: Poly C₁₂U), the new and improved form of Rugged dsRNA(e.g., Poly I: Poly C₃₀₋₃₅U (preferably, Poly I: Poly C₃₀U), whereinPolyC₃₀₋₃₅U, indicates a ratio, that is, that for every U there are30-35 C's), has an increased Ruggedness characterized by an increaseresistance to thermal denaturation and ribonuclease digestion. Thisimproved form of dsRNA also has a reduced tendency to form brancheddsRNA molecules which results in a increased bioactivity due to anincreased ability to bind TLR3 receptor. The minimal length of RuggeddsRNA (termed the monomer unit) is about 50 base pairs requiring about 4to 5 (e.g., 4.7) helical turns (10.7 base pairs are required for eachcomplete turn of the helix) within its dsRNA structure and representsthe smallest or monomeric unit of Poly I: Poly C₃₀U, approximately24,000 to 30,000 Daltons (a Dalton is a unit of weight equal to theweight of a single hydrogen atom). The maximal length of Rugged dsRNA isabout 500 base pairs composed of about 10 monomer units, requiring about50 (e.g., 46.7) helical turns and having a molecular weight ofapproximately 300,000 Daltons (e.g., about 225,000 Daltons). FIG. 21shows the relative size of the old unimproved Ampligen® vs. the Newimproved Rugged dsRNA.

Branched dsRNA

Branching is seen as the major configuration of dsRNA molecules withinthe Ampligen® unimproved mixture. FIGS. 26, 27, and 28 show typicalexamples of the branching molecules seen by Transmission ElectronMicroscopy (TEM) in the unimproved. Ampligen® mixture of dsRNAmolecules. The branching in the molecules interferes with TLR3 binding.Ampligen® has 4-5 times more molecules with ≧3 branched strands thanRugged dsRNA and, therefore, Ampligen® has reduced bioactivity relativeto the new and improved Rugged dsRNA (e.g., Poly I: Poly C₃₀₋₃₅U(preferably, Poly I: Poly C₃₀U)). This explains the increasedbioactivity of Rugged dsRNA compared to the unimproved Ampligen®mixture. Typical examples of the major component of Rugged dsRNA(unbranched dsRNA molecules) are shown in FIG. 29.

RNA Helix

A spiral structure of dsRNA with a repenting pattern described by twosimultaneous operations (rotation around the axis and translation alongthe axis). DsRNA regimes the translation of 10.7 base pairs to completeone complete rotation around the axis (i.e. one helical turn).

Dalton

A unit of weight equal to the weight of a single hydrogen atom. OneKda=1000 Daltons

Rugged dsRNA Monomer

The minimum active size of Rugged dsRNA comprised of about 40-50 basepairs, requiring about 4-5 (e.g., 4.7) helical turns and having amolecular weight of approximately 24,000 to 30,000 Daltons.

Rugged dsRNA Polymeric Units

Rugged dsRNA composed of multiple monomeric units (or fractional unitsthereof) of Rugged dsRNA up to a maximum of about 10 monomeric unitsheld together by covalent phosphodiester bonds in a linear structure.The maximum Rugged dsRNA molecular size contains about 500 base pairs,requiring about 50 (e.g., 46.7) helical turns and having a molecularweight of approximately 300,000 Daltons (e.g., about 225,000 Daltons).

Many uses of double-stranded ribonucleic acid (dsRNA) are known.Efficacy of such treatments, which includes a decrease in the numberand/or a reduction in the severity of adverse effects of nonselectedpopulations of dsRNA, is improved by the use of at least partiallypurified, Rugged dsRNA. The invention may be used to treat a subject(e.g., human or animal, especially birds, fishes, or mammals) with anincipient or established microbial infection, to treat a subject forother pathological conditions marked by abnormal cell proliferation(e.g., neoplasm or tumor), or for use as an immunostimulant to treat thesubject for a disease or other pathological condition caused by at leastinfection, abnormal cell proliferation, chronic fatigue syndrome, orcell damage from autoimmunity or neurodegeneration. It is preferred thatthe amount of Rugged dsRNA used is sufficient to bind Toll-Like Receptor3 (TLR3) on immune cells of the subject. Innate or adaptive immunity maybe triggered thereby. Preferably, Rugged dsRNA may be used to activateTLR3 selectively without activating other Toll-like receptors like TLR4or an RNA helicase like RIG-I or mda-5, or without inducing an excessivepro-inflammatory response as seen with the nonselective TLR3 agonistpoly (I):poly(C) in a phenomenon known as “cytokine storm” in the art.

The subject may be infected with at least one or more bacteria,protozoa, or viruses. A pharmaceutical composition which is comprised ofRugged dsRNA in an amount sufficient to bind to TLR3 is administered tothe subject. Infection of the subject is reduced or eliminated therebyas assayed by decreased reco-very time, increased immunity (e.g.,increase in antibody titer, lymphocyte proliferation, killing ofinfected cells, or natural killer cell activity), decreased division orgrowth of the microbe, or any combination thereof as compared to thesubject not treated with the Rugged dsRNA. The immunity induced bytreat-ment is preferably specific for the microbe, although inducinginnate immunity may also be efficacious.

An infection by a microbe may be treated. The microbe may infect a humanor animal subject. The infection may be incipient or established. Themicrobe may be a bacterium, protozoan, or virus; especially those thatcause disease (i.e., pathogenic microbes). Here, the terms “microbe” and“micro-organism” are used interchangeably.

The bacterium may be a species of the genus Bacillus (e.g., B.anthracis, B. cereus), Bartonella (B. henselae), Bordetella (e.g., B.pertussis), Bornelia (e.g., B. burgdorferi), Brucella (e.g., B.ebortus), Campylobacter (e.g., C. jejuni), Chlamydia (e.g., C.pneumoniae), Clostridium (e.g.., C. botulinum, C. difficile, C.perfringens, C. tetani), Corynbacterium (e.g., C. amycolatum, C.diphtheriae), Escherichia (e.g., E. coli O175:H7), Haemophilus (e.g., H.influenzae), Heliobacter (e.g., H. pylori), Klebsiella (K. pneumoniae),Legionella (e.g., L. pneumophila), Listeria (e.g., L. monocytogenes),Mycobacterium (e.g., M. avium, M. bovis, M. branderi, M. leprae, M.tuberculosis), Mycoplasma (e.g., M. genitalium, M. pneumoniae),Neisseria (e.g., N. gonorrheae, N. meningitidis), Pneumocystis (e.g., P.carinii), Pseudomonas (P. aeruginosa), Rickettsia (e.g., R. rickettsia,R. typhi), Salmonella (e.g., S. enteria), Shigella (e.g., S.dysenteriae), Staphylococcus (e.g., S. aureus, S. epidermidis),Streptococcus (e.g., S. pneumoniae, S. pyogenes), Treponema (e.g., T.pallidum), Vibrio (e.g., V. cholerae, V. vulnificus), or Yersinia (e.g.,Y. pestis). These include Gram-negative or Gram-positive bacteria,chlamydia, spirochetes, mycobacteria, and mycoplasmas.

The protozoan may be a species of the genus Cryptosporidium (e.g., C.hominis, C. parvum), Entamoeba (e.g., E. histolytica), Giardia (e.g.,(G. intestinalis, G. lamblia), Leishmania (e.g., L. amazonensis, L.braziliensi, L. donovani, L. mexicana, L. tropica), Plasmodium (e.g., P.falciparium, P. vivax), Toxoplasma (e.g., T. gondii), or Trypanosoma(e.g., T. bruci, T cruzi).

The virus may be a DNA or RNA virus that infects humans and animals, DNAviruses include those belonging to the Abenoviridae, Iridoviridae,Papiliomaviridae, Polyomavirididae, and Poxviridae families (Group Idouble-stranded DNA viruses); the Parvoviridae family (Group IIsingle-stranded DNA viruses). RNA viruses include those belonging to theBirnaviridae and Reoviridae families (Group III double-stranded RNAviruses); the Arteriviridae, Astroviridae, Caliciviridae, Hepeviridae,and Roniviridae families (Group IV positive single-stranded RNAviruses); and the Arenaviridae, Bornaviridae, Bunyaviridae, Filoviridae,Paramyxoviridae, and Rhabdoviridae families (Group V negativesingle-stranded RNA viruses). Rugged dsRNA may also be used to treatinfection by DNA viruses from the Herpesviridae family and RNA virusesfrom the Flaviviridae, Hepadnaviridae, Orthomyxoviridae, Picornaviridae,Retroviridae, and Togaviridae families.

The subject may be afflicted by a disease or pathological condition thatis characterized by abnormal cell proliferation (e.g., neoplasm ortumor, other transformed cells). A pharmaceutical composition which iscomprised of Rugged dsRNA in an amount sufficient to bind to TLR3 isadministered to the subject. Disease, symptoms thereof, their number, ortheir severity in the subject may be reduced or eliminated thereby asassayed by improved morbidity or morta-lity, increased immunity (e.g.,increase in antibody titer, lymphocyte proliferation, killingproliferating or transformed cells, or NK cell activity), decreaseddivision or growth of proliferating or transformed cells, or anycombination thereof as compared to the condition of a subject nottreated with Rugged dsRNA.

The subject's cells undergoing the abnormal proliferation may be aneoplasm or tumor (e.g., carcinoma, sarcoma, leukemia, lymphoma),especially cells transformed by a tumor virus (e.g., DNA or RNA viruscarrying a transforming gene or oncogene) or otherwise infected by avirus associated with cancer. For example, Epstein-Barr virus isassociated with nasopharyngeal cancer, Hodgkin's lymphoma, Burkitt'slymphoma, and other B lymphomas; human hepatitis B and C viruses (HBVand HCV) are associated with liver cancer; human herpesvirus 8 (HHV8) isassociated with Kaposi's sarcoma; human papillomaviruses (e.g., HPV6,HPV11, HPV16, HPV18, or combination thereof) are associated withcervical cancer, anal cancer, and genital warts; and humanT-lymphotrophic virus (HTLV) is associated with T-cell leukemia andlymphoma. Cancers include those originating from the gastrointestinal(e.g., esophagus, colon, intestine, ileum, rectum, anus, liver,pancreas, stomach), genitourinary (e.g., bladder, kidney, prostate),musculoskeletal, nervous, pulmonary (e.g., lung), or reproductive (e.g.,cervix, ovary, testicle) organ systems.

Dendric cell maturation may be induced in the subject. Immaturedendritic cells, which are capable of antigen uptake, may be induced todifferentiate into more mature dendritic cells, which are capable ofantigen presentation and priming an adaptive immune response (e.g.,antigen-specific T cells). During their conversion from immature tomature dendritic cells, they may at least change cell-surface expressionof major histocompatibility complex (MHC) molecules, costimulatorymolecules, adhesion molecules, or chemokine receptors; decrease antigenuptake; increase secretion of chemokines, cytokines, or proteases; growdendritic processes; reorganize their cytoskeleton; or any combinationthereof. They may be induced to migrate to sites of inflammation orlymphoid tissue through blood or lymph to bring microbes, neoplastic ortumor cells, or other transformed cells into proximity.

The subject may be vaccinated against at least infection or cancer. Insome cases, e.g., virus-induced cancers, both infection and cancer maybe treated. Immediately before, during, or immediately after vaccination(e.g., within 10 days of vaccination), a medicament or pharmaceuticalcomposition which is comprised of Rugged dsRNA in an amount sufficientto bind to TLR3 is administered to the subject. The immune response to avaccine or dendritic cell preparation is stimulated thereby. The vaccineor dendritic cell preparation may be comprised of killed, fixed, orattenuated whole microbes or cells (e.g., proliferating, ortransformed); a lysate or purified fraction of microbes or cells (e.g.,proliferating or transformed); one or more isolated microbial antigens(e.g., native, chemically synthesized, or recombinantly produced); orone or more isolated tumor antigens (e.g., native, chemicallysynthesized, or recombinantly produced). In situ vaccination may beaccomplished by the subject's production of antigen at a site orcirculation thereto (e.g., produced in a natural infection or cellgrowth, or shed antigen), and Rugged dsRNA acting as an adjuvantthereon.

Rugged dsRNA Rugged dsRNA

Rugged dsRNA as at least a portion of a medicament or formulated withother compatible components in a pharmaceutical composition may beadministered to a subject (e.g., human patient or animal, especiallybirds, fishes, or mammals) by any local or systemic route known in theart including enteral (e.g., oral, feeding tube, enema), topical (e.g.,device such as a nebulizer for inhalation through the respiratorysystem, skin patch acting epicutaneously or transdermally, suppositoryacting in the rectum or vagina), and parenteral (e.g., subcutaneous,intravenous, intramuscular, intradermal, or intraperitoneal injection;buccal, sublingual, or transmucosal; inhalation or instillationintranasally or intratracheally). The Rugged dsRNA may be micronized bymilling or grinding solid material, dissolved in a vehicle (e.g.,sterile buffered saline or water) for injection or instillation (e.g.,spray), topically applied, or encapsulated in a liposome or othercarrier for targeted delivery. Dissolving the Rugged dsRNA in water forinjection (WFI) and injection of the composition into the subject arepreferred. A carrier may be used to target the Rugged dsRNA to the TLR3receptor on antigen presenting calls and epithelium. For example,immature dendritic cells may be contacted in skin, mucosa, or lymphoidtissues. It will be appreciated that the preferred route may vary withthe age, condition, gender, or health status of the subject; the natureof disease or other pathological condition, including the number andseverity of symptoms; and the chosen active ingredient.

Formulations for administration (i.e., pharmaceutical compositions) mayinclude aqueous solutions, syrups, elixirs, powders, granules, tablets,and capsules which typically contain conventional excipients such asbinding agents, fillers, lubricants, disintegrants, wetting agents,suspending agents, emulsifying agents, preservatives, buffer salts,flavoring, coloring, and/or sweetening agents. It will be appreciatedthat the preferred formulation may vary with the age, condition, gender,or health status of the subject; the nature or disease or otherpathological condition, including the number and severity of symptoms;and the chosen active ingredient.

The recommended dosage of Rugged dsRNA will depend on the clinicalstatus of the subject and the physician's or veterinarian's experiencetreating the disease or other pathological condition. Rugged dsRNA maybe dosed at from about 0.5 μg to about 600 mg, from about 1 mg to about100 mg, or from about 10 mg to about 40 mg in a subject (e.g., body massof about 70-80 Kg for a human patient) on a schedule of once to thriceweekly (preferably twice weekly), albeit the dose amount and/orfrequency may be varied by the physician or veterinarian in response tothe subjects symptoms. Nucleic acid in solid form may be dissolved inphysiological phosphate-buffered saline and then infused intravenously.Cells or tissues that express TLR3 are preferred sites in the subjectfor delivering the nucleic acid, especially antigen presenting cells(e.g., dendritic cells, macrophages B lymphocytes) and endothelium(e.g., endothelial cells of the respiratory and gastric systems). Itwill be appreciated that the preferred dosage may vary with the age,condition, gender, or health status of the subject; the nature ofdisease or other pathological condition, including the number andseverity of symptoms; and the chosen active ingredient.

Dendritic cells which act as sentinel cells possess molecular surfacestructures that recognize pathogen-associated molecular patterns(PAMPs). These PAMPs include a set of Toll-like receptors (TLRs) thatspecifically recognize all dsRNA. In particular, dsRNA is a selectiveagonist of TLR3. Rugged dsRNA may be used as a selective agent toractivation of TLR3. Dysfunction in co-stimulatory molecule (e.g., CD80,CD83, CD86) signaling in dendritic cells may be associated with thedisease or other pathological condition to be treated. This abnormalitymay be normalized by using Rugged dsRNA as a selective TLR3 agonist. Theeffects of Rugged dsRNA may be inhibited or blocked by mutation of theTLR3 gene (e.g., deletion), down regulating its expression (e.g.,siRNA), binding with a competitor for TLR3's ligand-binding site (e.g.,neutralizing antibody) or a receptor antagonist, or interfering with adownstream component of the TLR3 signaling pathway (e.g., MyD88 orTRIF).

Circular dichroism (CD) is a physico-chemical technique forcharacterizing the conformation of specifically-configured dsRNA. It canalso be used as a surrogate for binding of Ampligen®dsRNA as a receptoragonist to its receptor TLR3. Furthermore, the helical structure ofRugged dsRNA and the structural requirements for binding of dsRNA toTLR3 can be precisely characterized by CD.

Other physico-chemical techniques that may be used to characterizeRugged dsRNA are reverse phase chromatography, PDA (photodiode array)analysis, gas pressure chromatography (GPC), specific ligand binding toTLR3 receptor, and sedimentation velocity measured byultracentrifugation.

Rugged dsRNA provides a selective agent for dissecting out the effectsof TLR3 activation on the immune system that was not previouslyavailable with such potency. Other agents like TLR adapters MyD88 andTRIF mediate signaling by all TLR or TLR3′TLR4, respectively. Thus,activation or inhibition of signaling through MyD88 or TRIF would notrestrict the biological effects to those mediated by TLR3. Since thepresence of TLR3 and its signaling is a requirement for Ampligen®poly(I):poly(C₁₂U) to act as a receptor agonist, one could assay for theabsence of TLR3 mutations, the presence of TLR3 protein, intactTLR3-mediated signaling, or any combination thereof in the cell ortissue of a subject prior to administration of the agonist. Suchconfirmation of TLR3 activity can be performed before, during, or afteradministration of the agonist. The agonist can be used to restrict theimmune response to activation of TLR3 without activating other Toll-likereceptors or RNA helicases. For example, abnormal cytokine (e.g., IFN-α,IFN-β, IFN-γ, TNF-α, IL-6, IL-10, IL-12) production or co-stimulatorymolecule (e.g., CD80, CD83, CD86) signaling may have resulted from atleast infection by the microbe, abnormal cell proliferation, autoimmunedamage, or neurodegene-ration. This abnormality may be remodulated byusing Rugged dsRNA as a selective agonist of TLR3. Antigen presentationmay be improved by conjuga-ting the antigen for a peptide analogthereof) to a ligand (or a receptor) that specifically binds to the cellsurface (especially a component of the endosome-phagosome internalizingpathway) of one or more antigen presenting cells. The specific bindingmolecule may be an antibody to a cell surface molecule, or a derivativethereof (e.g., Fab, scFv).

Expression of CD80, CD83, and CD86 may be analyzed by flow cytometryusing fluorescently-labeled antibodies. Following overnight shipment,blood samples are stained within one hour of receipt. Conventionaltechniques are used for lysis of red blood cells and cell markeranalyses by flow cytometry. Dendritic cells are identified based on lowlevel expression of lymphocyte monocyte, and NK cell markers along withhigh HLA-DR expression. Dendritic cells may also characterized accordingto CD11c and CD123 expression. Monocytes are identified by side scatteranalysis and expression of a monocyte lineage marker. Analyses of CD80,CD83, and CD86 expression are performed after cell type identification.Measurements from healthy volunteers serve as controls, and they wouldindicate normal distribution and levels of marker expression for maturedendritic cells such as CD80, CD83, and CD86.

Rugged dsRNA can be prepared by chromatographic separation, whereinRugged dsRNA is separated from the maturity of unimproved dsRNA.

An exemplary chromatographic procedure involves the following steps:

1. Binding of all forms of unimproved dsRNA to a reversed phasechromatography resin. The resin contains hydrophobic functional groups.In the current example the resin is Phenomenex, Polymerx, RP-1, but mayalternately be selected from a range of commercially availablehydrophobic resins as directed by conventional practice. Resin particlesize is 10 microns in the current example, but may be varied widely toafford optimal separations or to produce Rugged dsRNA at differingscales of operation.

2. The dsRNA is injected as a solution of 2.6 mg/ml in phosphatebuffered saline, pH 7.0, in the current example. The concentration andpH range can be varied to include alternative appropriate bufferingsystems utilized by those familiar with the art. The diluent can alsocontain stabilizing elements such as magnesium. The ionic strength, 200mM in the current example, can be varied to achieve optimal loading andseparation performance conditions as directed by conventional practices.

3. The mobile phase composition contains a relatively poser organicsolvent to modulate binding during loading and the subsequent, gradientelution. In the current example, the mobile phase contains acetonitrileat an initial loading concentration of 6-8 vol % which produces enrichedRugged dsRNA fractions during the gradient elution to 20 volume %.Alternative solvent systems can be selected having optimal solventconcentration ranges as directed by conventional practice.

4. The sample loading is 13 mg unimproved dsRNA/ml column. Loading canbe decreased to afford tighter fractionation of Rugged dsRNA.Alternative combinations of solvent and ionic strength will requireindividually determined optimal loading conditions as directed byconventional practice.

5. The mobile phase flow rate for sample loading and elution range is 5ml/min in the current example (3 column volumes/hr). The flow rate canbe varied to achieve optimal conditions for differing scales ofoperation and resin particle size as directed by conventional practice.

8. Elution is achieved by imposing a solvent gradient to the compositionof the mobile phase. In the current example, the gradient ofacetonitrile composition is increased from the loading condition of 6-8%to 20%, over a period of 14 minutes. The type of solvent and thegradient profile can be altered based upon the character of thehydrophobic functionality as determined by conventional practices.

7. In the current example, improved Rugged dsRNA is collected at 10-12minutes or 0.25-0.30 column volumes. The peak location can varydepending upon alternative choices of solvent, flow rate, column type asprovided above.

One skilled in the art will appreciate that separations suitable forisolation of Rugged dsRNA can be scaled up for commercial purposes.

EXAMPLES

Synthesis of single-stranded poly(I) and poly(C₁₂U) began withenzymatic, polynucleotide synthesis of the polynucleotides from therespective mononucleotide starting materials: inosine for poly(I);cytidine (C) and uridine (U) for poly(C₁₂U). Then repetitive extractionand precipitation steps were used to remove residual impurities. Thereaction solutions containing the products were concentrated byultrafiltration and extracted with phenol four times. The concentratedand extracted solutions were precipitated, dissolved, and reprecipitatedfrom aqueous ethanol (50:50). Whereas precipitated poly(I) was separatesby centrifugation, the supernatant (waste) liquid phase of adherentpoly(C₁₂U) was simply removed by aspiration. The precipitated pasteswere redissolved, then concentrated, diafiltered, and furtherconcentrated. The final bulk solutions containing polynucleotide wasfiltered. The filtered solution was freeze dried and the raw materialswere stored frozen.

Enzymatic Synthesis. The enzymatic synthesis used in the manufacturingprocess is dependent on the enzyme polynucleotide phosphorylase tosynthesize polyinosinic acid and polycytidilic₁₂uridilic acid from theirrespective starting materials: cytidine 5′-diphosphate, trisodium salt(CDP.Na₃), uridine 5′-diphosphate, disodium salt (UDP.Na₂) and inosine5′diphosphate, trisodium salt (IDP.Na₃).

The enzyme catalyzes polynucleotide formation in a reversible reactionusing Mg⁺⁺ as a co-factor and ATP as a source of energy. Polynucleotideswere synthesized in the 5′ to 3′ direction with concurrent liberation ofinorganic phosphate. Maximum yield was limited by the equilibriumbetween synthesis and reverse rates, degradative reaction(phosphorolysis). The progress of the reaction was followed by measuringthe consumption of CDP or IDP. Viscosity of the reaction solution wasalso monitored. Purified water was filtered into the tank. The followingingredients were added to the tank one at a time with mixing: TRIS(hydroxymethyl) aminomethane, urea, magnesium chloride hexahydrate(MgCl.6H₂O), and ethylenediaminetetraacetic acid (edetate), disodiumsalt (EDTA.Na₂). Raw material mononucleotides were also added.

Each ingredient was dissolved before the next one was added. After allof the ingredients were added, the solution was mixed for a minimum of10 minutes. The mixture was then adjusted and purified wafer was addedto obtain a final batch volume. This pre-enzyme reaction mixture wassampled tor initial CDP or IDP concentration. The enzyme polynucleotidephosphorylase was added with mixing, whereupon the synthesis ofpolynucleotide commenced. Also, the viscosity profile at the optimalenzyme concentration must exhibit the usual increase in viscosity overtime without significant decrease at the conclusion of the batchreaction; significant decrease in viscosity would indicate undesireddegradation of polynucleotide. After the optimized amount of enzyme wasadded to the production batch, enzymatic synthesis progressed underconstant, controlled agitation. The consumption of CDP or IDP wasmonitored approximately every hour. The reaction was terminated by theaddition of a stop solution. Viscosity was also monitored, forinformation only, during the process.

Concentration of Reaction Solution. To minimize the required volume ofphenol for extractions, the reaction product solution was concentrated.

Extraction of dsRNA mixture. Residual enzyme was removed predominatelyby phenol extraction. The concentrated single stranded RNA reactionproduct solutions were transferred into seperate extraction tanks and 2MTRIS and sodium dodecyl sulfate (SDS) were added. After at least 5minutes of mixing, liquefied phenol was added and the two phase solutionwas mixed to disperse the phenol phase in the aqueous phase. SDS wasemployed as a surface-active agent to facilitate dissolution ofdenatured protein into the phenol phase; TRIS was required to buffer thesolution at an optimal pH for polynucleotide stability. The extractionmixture stands without mixing for predetermined settling times to affordcoalescence of the phases. The lower phenol waste phase is then pumpedinto containers for disposal. The location of the phenol cut wasimportant in order to effectively separate phenol and protein from theupper, product phase, which contains single stranded RNAs. The phenolphase and an intermediate “rag” layer, which contains denatured proteinsolids, were discarded by visually observing the liquid flowing throughthe site glass at the tank outlet. When the phenol and rag layerdisappeared and only product phase was observed, the outlet valve wasclosed and the phenol cut is consi-dered complete.

Precipitation of single stranded RNAs. Contaminating phenol, SDS, andother salts remaining in solution were removed by precipitation withdenatured ethyl alcohol. The single stranded RNA concentrated solutionwas pumped into the precipitation tank. The denatured alcohol was addedand after mixing the precipitate was separated.

Concentration and Diafiltration. Remaining bulk salts, a small amount ofunreacted mononucleotide, and phenol were removed by diafiltrationagainst water. The precipitate was dissolved in the originalprecipitation vessel with gentle mixing and heating. After dissolving,the solution was then concentrated and diafiltered against water forinjection (WFI). The solution was filtered prior to freeze drying.

Freeze Drying. The filtered single stranded RNA material was loaded intoa freeze drier. The material was frozen, and a vacuum was then applied.The product was considered dry when the programmed cycle was complete.

Manufacture of dsRNA, Sterile Solution, for Intravenous Infusion. Thesingle stranded RNAs were dissolved in phosphate-buffered saline. Equalmolar amounts were mixed in an annealing step, and cooled to roomtemperature. The solutions were sterile filtered.

Preparation of Buffer Vehicle, Excipient Solution. WFI was added to thetank. The excipients were added to the tank, and mixed. After mixing,the batch was sampled for pH and osmolality. Quality control must bewithin in-processs limits prior to use for formulating the solutions.

Formulating Poly(I) and Poly(C₁₂U) solutions. An initial quantity ofbuffer solution was subdivided according to the batch formula and wasfiltered into the tank. The single amended RNAs were added to the buffersolution, and dissolved by mixing. The temperature of the solution wasincreased and maintained with mixing. The solution is then recirculated.

Annealing of Poly I:Poly C₁₂U Strands. Equivalent quantities of poly(I)and poly(C₁₂U) were transferred to the tank. With continual mixing, thetemperature of the solution was increased. Samples were removed andtested for potency, and pH.

Sterile Filtration. The formulated bulk was sterile filtered inclineinto a steam sterilized surge vessel.

Filling Operations. The filling operation was performed. After each vialwas filled, a sterile stopper is used to stopper the vial. Stopperedvials were then conveyed from the aseptic processing area where theywere sealed.

Rugged dsRNA was isolated from the annealed poly(I):poly(C₁₂U) mixture,which was prepared according to the above, by either analytical orpreparative high performance liquid chromatography (HPLC) as asubstantially purified and pharmaceutically-active molecule. Itsmolecular weight is from about 30 Kda to 300 Kda and is about 50 to 300base pairs in length with about 4.7 to 46.7 complete turns of the RNAhelix. It is only from about 4 mol % to about 16 mol % of anunfractionated Ampligen® composition. Most dsRNA (over 80 mol %molecules) after synthesis have a molecular weight of about 1.2 Mda andare about 2000 base pairs in length with about 187 complete turns of theRNA helix. The Rugged dsRNA in the 5 min HPLC peak is about 4 to 40times smaller than the bulk of the dsRNA, and more closely fits theligand binding site of its cell surface receptor (TLR3).

Due to its structure, Rugged dsRNA is unusually resistant to disruptionor its RNA double helix and molecular unfolding. Thus, Rugged dsRNAunder the essay conditions described herein has about 100- to about1,000% greater bioactivity than the same weight of unimproved Ampligen®poly(I):poly(C₁₂₃U).

(a) Protection by Mismatched dsRNA is by Selective Activation of TLR3

TLR3 Activation A Linked to Expression of IFN-α/β, IL-6, or IL-12. Therelationship between IFN expression through TLR3 activation by dsRNA wasestablished by Alexopoulou (2001) using 293T cells that expressdifferent Toll-like receptors (human TLR1, TLR2, TLR3, TLR4, TLR5, TLR6,or TLR9). Only those cells containing human TLR3 showed markedexpression of IFN-α/β, IL-6 or IL-12 when stimulated with poly(I):poly(C).

Mismatched dsRNA induces Host Defense Gene Modulation through HighlySelective Activation of TLR3. To understand the relationship of theTLR3-dependent innate immune response to viral protection, Gowen (2007),subjected TLR3-deficient mice to dsRNA and measured expression ofIFN-α/β, IL-6, and IL-12. The mice were also subsequently challenged byexposure to Punta Toro virus (PTV). Protection from the viral challengewas exquisitely sensitive to treatment with mismatched dsRNA. Viralprotection conferred by mismatched dsRNA was completely abolished forthe case of TLR3-deficient mice. When contrasted to the partial butsignificant effectiveness of mismatched dsRNA in TLR3^(−/−) mice, it isclear that the structural substitutions of uridine in the cytidinestrand of mismatched dsRNA are responsible for the highly specific,TLR3-dependent pathway. Furthermore, measurements of IFN-α/β and IL-6directly link PTV protection or lack thereof to the modulation of thesecytokines.

This selective targeting of the TLR3 signaling pathway represents asignificant advantage for therapeutic applications of mismatched dsRNAas compared to other possible cytosolic mechanisms such as, for example,the use of unsubstituted dsRNA poly(I):poly(C) to stimulate cytokineproduction through RNA helicases such as MDA-5 and RIG-1 (Pichlmair,2006).

(b) Binding of dsRNA to TLR3 Requires a Helical Non-BranchingConformation of dsRNA

Molecular Model of the human an TLR3 dimer ecodomain and its RuggeddsRNA ligand. The X-ray crystallographic structure of the mouse TLR3dimer/dsRNA complex (3CIY) and the human TLR3 monomer (1ZIW) providedthe coordinates for the model of human TLR3 with Rugged dsRHAbound toits active site (Liu, 2008; Bell, 2005). Molecular modeling usedAccelrys' Discovery Suite software (version 2.5.5). The dsRNA structureof 3CIY was mutated in situ to the respective poly I and poly C₃₀Uchains (Rugged dsRNA) maintaining the phosphate backbone lineartranslational coordinates of the X-ray crystallographic structure thatis represented as lines for its structure. A uridine in the poly C₃₀Ustrand is at position U23. The coordinates of the human TLR3 monomerwere used to replace each homodimer. Several unacceptable close van derWaals contacts for the human TLR3 crystal coordinates were resolved byan alternate rotamer conformation of the individual amino acid R group.The TLR3 homodimers are represented as structural elements with the bluearrows signifying direction of β-sheets and the red cylinders signifyingα-helices. FIG. 24 is viewed from a lateral view of Rugged dsRNA boundto the active site of the TLR3 homodimer. The C-terminal regions of eachdimer face each other and bind to the phosphate backbone of the dsRNA.The N-terminals of each TLR3 bind to opposite ends of the dsRNA with aminimum length of 50 bp required for interaction with essential residuesof TLR3 for activation of intracellular signaling. Amino acids of TLR3required for binding of Rugged dsRNA are shown as Van der Waals' radiiassociated with the phosphate backbone. FIG. 25 illustrates the TLR3homodimer complexed with Rugged dsRNAas seen down the long axis of thedsRNA.

TLR3 Binding Site. Studying the structure of native TLR3 crystals. Choe(2005) found that TLR3 is a large horseshoe-shaped, right-handed,solenoid structure comprised of 23 leucine-rich repeats. Theglycosylated, convex surface and negatively-charged concave surfaces areunlikely binding sites for dsRNA. Consequently, they proposed thatdsRNA-binding occurs at positively-charged patches located on thelateral face.

Using mutational analysis, Boll (2005, 2006) modified putative TLR3binding sites in the positively-charged patches and observed formationof a dsRNA/TLR3 complex by size-exclusion chromatography. Despite thepresence of numerous positively-charged residues, only two amino acidsN541 and H539 were required for binding. The amido group of H539 couldinteract with dsRNA by hydrogen binding. Proximity of the secondpositively-charged residue N541 was also important, albeit the role ofthis amino acid was not as clear. Mutation to negatively-chargedaspirate prevented binding by dsRNA, however conversion to a neutralalanine residue had no effect on binding by dsRNA.

Binding to TLR3 Requires Helical Conformation of dsRNA. Following thestructural determination of most likely dsRNA binding surfaces on TLR3.Choe (2005) further proposed that the helical symmetry of dsRNAstructure is necessary for the creation of the summetric dimer form ofactivated, membrane-associated TLR3. In the ternary complex at themembrane surface, two symmetrically opposed TLR3 molecules are linked toeither side of the helix of the common dsRNA.

As discussed above, using mutational analysis, Bell (2005, 2006) definedtwo highly conserved residues (N541 and H539) that are necessary forbinding of dsRNA to TLR3. Moreover, the constraining requirement forligand binding to both of these residues of TLR3 is satisfied only bythe minor groove architecture of the (helical) conformation of dsRNA.When dsRNA phosphate binds in proximity to the charge sensitive H539,then the amide side (H541) becomes aligned with hydrogen bonding site ofa 2′ dsRNA hydroxyl only when helical dimensions are utilized.

(c) Helical Conformation of dsRNA and Alteration Thereto AccompanyingLigand Interactions are Precisely Characterized by Circular Dichroism

Circular dichroism provides detailed information concerning thesecondary, helical structures of dsRNA or alterations thereof whichaccompany ligand binding; as well as structural changes caused byenzymatic hydrolysis and addition of metal ions. Also, in the thermalstress mode conformational information imparted by CD provides valuableinsights to explain RNA stability.

dsRNA Characterization. Gray (1995) showed that CD, applied in themixing curve protocol, complemented ultraviolet absorption measurementsto determine the stoichiometry of duplex RNA (A-G; C-T(U)). In thisapproach, the optical property is analyzed as a function of the addedratios of individual strands. The magnitudes of CD difference plots weremaximal for 50:50 mixtures. Further, isodichroic behavior correlatedwith the formation of higher ordered or intra strand structures.

Ligand Interactions. Ghazaryan (2006) studied the ligand interaction ofdsRNA with a family of positively charged pyridinium porphyrins. From CDmeasurements they found that minor modifications of porphyrin structureled to profound differences in mode of their attachment to the doublehelical structure. Whereas TEtOHPyP4 associated by intercalation,TMetAlPyP4 attached by forming an external, self-stacking assembly.

Using circular dichroism. Brown (2002) showed that ADAR1, a human dsRNA,(chimeric) converted from the A to Zα form upon binding to adenosinedeaminase. Corroboration was provided by crystallization of the complexand Raman spectroscopy. Sorrentino (2003) studied the powerful enzymaticdegradation of dsRNA by human pancreateic ribonuclease (HP-RNase).Circular dichroism of the RNA/enzyme complex repealed that multi-siteattachment or the dsRNA to HP-RNase was responsible for thedestabilization of the RNA helix.

Stability of dsRNA. Studying the rRNA component of the 70S ribosomalcomplex, Sumita (2005) showed that pseudouridine substitutionsstabilized the dsRNA helix based upon structural information provided bycircular dichroism (CD). Specifically, pseudouridine substitutionscreated duplex regions with closing base pairs and water-mediatedhydrogen bonds. Stabilization by Mg⁺⁺ was also characterized by CD inthis study. Investigating the stability of RNA-DNA hybrids with variantsin base composition, Lesnik (1995) showed that more stable hybridsretain ellipticity at 210 nm a wavelength characteristic of the singlecomponent RNA band (A-form hybrid). In contrast, less stable hybridsshowed lowered 210 nm ellipticity, values which wore intermediatebetween the RNA and DNA components.

A double-stranded RNA composition may be analyzed by high performanceliquid chromatography (HPLC) an shown in FIGS. 1A, 1B and 1C. Analysisof a representative lot of Ampligen® poly(I):poly(C₁₂U) mixture resultedin two distinct peaks: one with retention times from 9.85 to 10.35 mincorresponding to the poly(I) strand and from 7.30 to 7.80 mincorresponding to the poly(C₁₂U) strand. Rugged dsRNA is found at aretention time of about 5 min representing a molecular species uniquelyresistant to denaturation and unfolding. Denaturing conditions wouldeliminate biological activity exclusively due to TLR3 receptor binding.This analytical method may also be used as a stability indicating assayand, in particular, it may be used to show that the Rugged dsRNA isunusually resistant to disruption of its double helix and to molecularunfolding.

For the lyophilized (freeze-dried) preparation (FIG. 1B), aggregatesmaybe present and elute at 13 and 15 min. A small fraction of individualnucleosides, inosine, cytosine and uridine, elute at 1 min. The overallpurity of poly I:polyC₁₃U (Ampligen®) determined by HPLC is representedby the sum of the 7.4, 8.7 and 10 minute peaks and is 96-99%. “NewdsRNA” comprises 1-4% and is different from Ampligen® by its size andphysico-chemical properties as discussed herein.

The identity of each peak is determined by analysis with a photodiodearray (PDA) detector as shown in FIGS. 2A, 2B and 2C. At each selectedretention time, a UV absorption scan of wavelengths from 200 nm to 360nm was obtained. Duplex poly(I):poly(C₁₂U) and individual poly(I) andpoly(C₁₂U) strands have their own specific peak absorption wavelengths.Absorption peaks centered at note 248 nm and 265 nm indicate thepresence of Rugged dsRNA (about 286,000 daltons) having poly(I) andpoly(C₁₂U), respectively (FIG. 2A). Peak absorption centered at about265 nm indicates the presence of the poly(C₁₂U) strand (FIG. 2B). Peakabsorption centered at about 248 nm indicates the presence of thepoly(I) strand (FIG. 2C). Absorption centered at about 230 nm is due toacetonitrile used as solvent. Because of the relative scarcity of RuggeddsRNA, the signal at 230 nm was subtracted from FIG. 2A.

FIG. 21 shows the relative size of Ampligen® vs new Rugged dsRNA (peak 5minutes)

Shown in FIG. 22 is a partial view of poly(I):poly(C₁₂U) partiallyhybridized strands and the interaction of bases of individual poly(I)and the poly(C₁₂U) strands. Single inosine bases bind to cytosine bases,but not to the uridine base. In this structure, the poly (inosinic acid)is hydrogen bonded (dashed lines between bases) to poly (cytidylicacid), with uridylic acid substitution occurring on an average of every12-13 bases.

-   -   Molecular formula:        (13C₁₀H₁₁N₄O₇P)_(n):((12C₉H₁₂N₃O₇P)(C₉H₁₁N₂O₈P))_(n)    -   Molecular size: about 1,200,000 daltons        The number of repeat units (n) corresponding to the size of        poly(I):poly(C₁₂U) of approximately 1.2 Mda is 2000 base pairs        or 187 full helical turns.

TABLE 2 Molecular Weight (MW) of Unimproved Ampligen ® MixtureComponents Common name: poly(I):poly(C₁₂U) (1,200,000 daltons) Chemicalname: poly(inosinic acid):poly((cytidylic acid)₁₂(uridylic acid)) MWUnit Unit MW* Inosine 5′ mono- 330 13 4056 phosphate Cytidine 3′ mono-305 12 3444 phosphate Uridylic acid 306 1 288 Overall Average: 318 Sum:7788 *Note: One molecule of H₂O(mw = 18)is lost for each phosphodiesterbond formed

Shown in FIG. 23 is a partial view of Rugged dsRNA, poly(I):poly(C₃₀U),partially hybridized strands and the interaction of bases of individualpoly(I) and the poly(C₃₀U) strands. Single inosine bases bind tocytosine bases, but not to the uridine base. In this structure, the poly(inosinic acid) is hydrogen bonded (dashed lines between bases) to poly(cytildylic acid), with uridylic acid substitution occurring on anaverage of every 30-31 bases. This is “rugged” dsRNA.

-   -   Molecular formula:        (31C₁₀H₁₁N₄O₇P_(n):((30C₉H₁₂N₃O₇P)(C₉H₁₁N₂O₈P))_(n)    -   Molecular size: about 300,000 daltons

The number of repeat units (n) corresponding to the size range of newvariant, also termed Rugged dsRNA (also termed peak 5 min on HPLC) isabout 30-300 Kda having about 50-500 base pairs representing 4.7-46.7complete turns of RNA helix and is resistant to disassembly ofhydrogen-bonded strands under elevated thermal or abnormal ionicconditions.

TABLE 3 Molecular Weight (MW) of Novel Rugged dsRNA Components. MW UnitUnit MW* Inosine 5′ mono- 330 31 9,672 phosphate Cytidine 3′ mono- 30530 8,610 phosphate Uridylic acid 306 1 288 Overall Average: 318 Sum:18,570 *Note: One molecule of H₂O(mw = 18)is lost for eachphosphodiester bond formed

Circular dichroism (CD) has bean used to measure secondary structure(duplexed helices) of biological and synthetic polymers, includingproteins and nucleic acids. CD is the measurement of absorption ofright- or left-circular polarized light, at a specific wavelength, bychiral molecules. Chemical chirality is the property of a molecule beingnonsuperimposable on its mirror image. An atom that makes its moleculechiral is called a chiral atom or, more commonly, a chiral center.Rugged dsRNA and Poly(I):poly(C₁₂U) have a number of chiral centersbecause of their primary and secondary structures. Chiral centers arefound in the nucleotide bases, which term the two primary structures forthe two individual RNA strands (ssRNA) of Rugged dsRNA andpoly(I):poly(C₁₂U). Additional chiral centers some from hybridizing eachssRNA to the other through hydrogen bonding of their complementarybases. Hydrophobic bonding between adjacent bases of dsRNA is known asbase stacking and produces a flexible, linear symmetrical, helicalsecondary structure of defined shape and size. CD Spectra for RuggeddsRNA and Ampligen®, which are dependent on the wavelength, are observedto be a function reflecting the Gaussian absorption for each chiralcenter. Therefore, the CD spectrum for a dsRNA such as Rugged dsRNA isdependent on the complementary base pairing of double-strandedstructures and the complex chirality of the resultant helical structure.

It has been demonstrated by UV and CD spectroscopy that the biologicalactivity of dsRNA is dependent on these specific spatial and stericconfigurations. Since perturbation of helical structure results in lossof the chiral centers characteristic of the secondary structure, theanalysis and monitoring of secondary structure by CD provides a methodto characterize the physico-chemical properties of Rugged dsRNA andpoly(I):poly(C₁₂U) that are associated with their bioactivity.

The specific ellipticity measured in a wavelength scan provides aquantitative parameter, which is calculated as the ellipticity ratio atcertain “critical” wavelengths. The value of this structural parameter,the ratio CD₂₇₈/CD₂₄₅, is a characteristic of Rugged dsRNA or theunimproved Ampligen® mixture. In a second CD analysis, ellipticity ismeasured during heating. As poly(I):poly(C₁₂U) is heated and thermallydenatured, the individual poly(I) and poly(C₁₂U) strands unwind due tothe breakdown of hydrogen bonding between complementary base pairs. Whenthe temperature derivative of ellipticity is plotted, the minimumderivative value corresponds to melting temperature, defined as thepoint where 50% of the double-stranded conformation is unwound. Thewidth at half-height of the peak, a measure of structural uniformity,also becomes an indication of its integrity. Taken together, thesethermal indices provide a measure of the strength of the dsRNA helixes.

The wavelength scan detects two peaks: a first peak at 245 nmcorresponding to the doubled stranded helix of the poly(I):poly(C₁₂U)and a second peak at 278 nm corresponding to the stacking of the nucleicacid's base pairs. As shown previously in FIGS. 4 and 5, dsRNA affordsseparate peaks in the CD wavelength scan, at 245 and 278 nm, the formerpeak associated with base stacking attribute of helical structure.Accordingly, the ratio of peak heights at 278/245 is typically within0.69-0.79 for dsRNA out much higher in the absence of double helicalstructure.

Table 4 summarizes CD wavelength scans obtained by isolation offractions of the three reversed phase HPLC peaks previously discussed inFIGS. 1B and 1C. The reversed phase HPLC assay is utilized todistinguish “rugged” dsRNA (5.0 minute peak) from the separated,component strands of poly(I):poly(C₁₂U): 7.0 (poly C₁₂U) and 10.0 minutepeaks (poly I). It is clear from the 278/245 ratio that only the 5.0minute, Rugged dsRNA fraction retains helical structure, in contrast tothe separated, component strands of Poly I: Poly C₁₂U.

This result underscores the greater stability of Rugged dsRNA during thereversed phase isolation, in which all polynucleotides experiencebinding and elution. Whereas poly(I):poly(C₁₂U) is separated into thecomponent 7 and 10 minute polynucleotide strands, the 5 minute. RuggeddsRNA, retains the double stranded conformation.

TABLE 4 PREPARATIVE HPLC*: Peak Analysis by Circular Dichroism PeakCircular Dichroism Lot (min) 278/245 nm response requirement (0.69-0.79)0303SD 5 Min 0.78 7 min 12.3 10 min  1.74 0301SD 5 Min 0.79 7 min 2.4910 min  N/A *Preparative HPLC Methodology:

-   HPLC Equipment: Beckman Coulter Preparative HPLC (System. Gold 126P    Solvent Module), Bookman coulter (System Gold 168 Detector)-   Column: Phenomenex, Polymerx, 10μ, RP-1, 100° A, 250×21.20 mm-   Mobile Phase: 200 mM of Triethylamine Acetate buffer, pH 8.7-   Flow Rate: 5.0 mL/minute-   Infection Volume: 5 mL-   Wavelength: 255 nm and photodiode array detection.

TABLE 5 Gradient Condition: Time (Minute) Acetonitrile (%) Buffer (%) 08 92 3 10 90 6 12 88 9 14 86 12 16 84 14 20 80 30 20 80The column was equilibrated with 8% acetonitrile and 92% buffer for atleast 30 minutes. Peaks were collected at 10-12 minutes and 22-27minutes which corresponded to differing gradient acetonitrilecompositions indicated in Table 5 above. The injection process wasrepeated 20-30 times and fractions from the first peak (10-12 minutes)were pooled for subsequent analysis.The pooled fractions were concentrated and solvent was displaced with awater wash, using Amicon Ultra Centrifugal Filters (Amicon Ultra, CatUFC 50104). The concentrated samples were analyzed for concentration byUV, based upon averaging of the concentration responses which wereseparately calculated at the wavelength maximum for each polynucleotidechain. ε=5.2×10̂3 at λ=265 nm and ε₂=4.9×10̂3 at λ=249 nm.Only 5 minute exhibits double stranded base stacking character:

-   -   Significant 245 nm response reflecting double strand helix (245        nm)    -   Acceptable 245/278 Ratio reflecting base pairs=278 nm (chiral        centers in backbone).

Precision, Ampligen® poly(I):poly(C₁₂U), lot 9807CD, at a concentrationof 2.5 mg/mL was repeatedly assayed to investigate the precision of theCD assay. The percent relative standard deviations (% RSD) for themelting temperature (T_(M)), for the width at half-height for the firstderivative of the melting curve and for the ratio of measurements of theCD peaks at 278 nm and 245 nm were calculated as 0.76%, 9.09%, and1.41%, respectively. This demonstrated that CD assay of Ampligen®poly(I):poly(C₁₂J) acts in a precise manner during thermal analysis forthe determination of T_(M) and width at half height of the firstderivative of the thermal melt curve arid during the CD scan analysisfor determination of the ratio of CD at 278 nm to CD at 245 nm.

Specificity. This CD method for characterizing poly(I):poly(C₁₂U) isalso specific because it can between differentiate duplexes nucleicacids and single-stranded nucleic acids, or other similardouble-stranded nucleic acids that do not meet the manufacturing andrelease specifications, for Ampligen® poly(I)-poly(C₁₂). The specificityof this method, in regards to analysis of single versus double-strandednucleic acids, was demonstrated by comparing scanning profiles andmelting temperature curses. See FIG. 7. The scans of double-strandedmolecules such as poly(I):poly(C₁₂U), poly(I):poly(C), andpoly(A):poly(U) differed significantly from those obtained duringanalysis of single-stranded molecules such as poly(I) and poly(C₁₂U).See FIGS. 8-17. Furthermore, each of the CD scans was unique for themolecular species being assayed.

The scecificity of the assay was also investigated to assess,unequivocally, the ability to detect compounds of closely relatedstructure.

(a) Double-stranded ribonucleic acids of different nucleotide basecomposition, such as poly(I):poly(C₁₂U), poly(I):poly(C), andpoly(A):poly(U). FIGS. 10, 12 and 14).(b) Ampligen® poly(I):poly(C₁₂U) that meets the polymer sizespecification.(c) Double-stranded ribonucleic acid formulated from poly(I) andpoly(C_(x)U_(y)) strands with a cytidine to uridine base ratio of 11-14to 1 (FIGS. 16 and 17) (C:U ratio=11:1 to 14:1).

The specificity of assays for dsRNA that differed in their nucleotidebase composition was evidenced by comparison of CD scans and meltingcurves of similar, but different, double-stranded molecules, such aspoly(I):poly(C₁₂U), poly(I):poly(C), and poly(A):poly(U). CD scanningprofiles appear to be similar, as seen with the scans ofAmpligen®poly(I):poly(C₁₂U) and poly(I):poly(C). But calculations of theratios obtained at 278 nm and 245 nm, and subsequent t-test statisticalanalysis for equal means showed that the CD scan of Ampligen® differssignificantly from similar dsRNA having different nucleotide basecompositions. Specificity for the dsRNA of different nucleus acid basecomposition was also demonstrated by their thermal melting curves.Thermal melt curves for dsRNA differed significantly from each other.Statistical analysis (t-test for equal means) of data from the plots ofthe first derivative of the availing carves confirmed mat the resultsobtained for their respective T_(M) and width at half-height aresignificantly different. Therefore, specificity of the CD methoddifferentiates Ampligen® from other dsRNA mole-cules by parameters ofboth the scan and the thermal melt profiles.

The CD method is specific for detection of poly(I):poly(C₁₂U) formulatedfrom polymers not meeting the aforementioned specifications for size.When one or both polymers of the poly(I):poly(C₁₂U) molecule is outsidethe 4-8S size specification, the results from the CD analysis of thesemolecules do not meet specifications for Ampligen® in regards to T_(M)and width at half-height of the first derivative of the thermal meltcurve. The failure to meet speci-fications for these CD parameters isobserved with these formulations even when the ±1.5S size differentialspecification is satisfied. Relative to the data obtained from thethermal melt analyses of Ampligen® formula-tions, the CD₂₇₈/CD₂₄₅ ratiodeterminations were leas specific. CD scans alone did not differentiatebetween poly(I):poly(C₁₂U) and non-poly(I):poly(C₁₂U) formulations thatdid not meet manufacturing and/or release specifications for polymersize.

As discussed above, the specificity of CD analysis is sensitive to thesize of the single-stranded polymer strands. In addition, when the sizedifference between the complementary single-stranded polymer components,poly(I) and poly(C₁₂U), is 2.4S or greater, the CD thermal melt analyseswill differentiate poly(I):poly(C₁₂U) from similar molecules not meetingthe specification for the complementary polymer size differential.

CD analysis can distinguish between poly(I):poly(C₁₂U) and similarmolecules that do not meet specifications for the amount of doublestrandedness or base pairing between the complementary poly(I) andpoly(C₁₂U) strands. The amount of base pairing is dependent on therelative proportion of cytidylic acid to uridylic and (C:U ratio) of thepoly(C_(x)U_(y)) polymer. The ratio of cytidine to uridine in thepoly(C_(x)U_(y)) polymer affects the melting temperature (T_(M)) as wellas the width at half height of the first derivative of the meltingcurve. When the ratio of cytidine to uridine is less than 11:1, there isless double standedness or base pairing (between polyinosinic acid andpolycytidylic acid complementary strands of the duplex RNA helix) thanthat for Ampligen®. This results in lower observed T_(M)'s and largerwidths at half-height for the first derivative of the thermal meltcurves relative to those observed for poly(I):poly(C₁₂U). Increasing thecytidine to uridine ratio of the poly(C_(x)U_(y)) strand increases thebase pairing between the complementary strands of the helix and,therefore, increases the observed T_(M) and decreases the observedwidth, at half-height of the first derivative of the thermal curve. TheCD₂₇₈/CD₂₄₅ ratio determi-nations were demonstrated to be less sensitiveto differences in the C:U ratio in Ampligen® formulations.

Both the size of the complementary polymer strands and the C:U ratio ofthe poly(C₁₂U) strand contribute to double strandedness of apoly(I):poly(C₁₂U) molecule. The double strandedness, in turn,contributes to the efficacy of the drug product as discussed in theintroduction. Therefore, CD method is an important analytical tool forcharacterization of poly(I):poly(C₁₂U). Although CD scans anddeterminations of the CD₂₇₈/CD₂₄₅ ratio are less specific than thethermal melt analysis determinations of T_(M) and width at half-heightof the first derivative of the melt curve, all three CD parameters maybe used in combination for the thorough characterization andidentification of poly(I):poly(C₁₂U).

Bioactivity and Stability of Rugged dsRNA

Bioactivity of dsRNA and poly(I):poly(C₁₂U) were measured, and thancompared utilizing a ligand-binding assay. Stability was measured usingthe product release test, reverse phase HPLC assay.

A summary of the results is presented below, followed by more detaileddiscussion. The combination of enhanced bioactivity and much greaterstability under the thermal stress of 40° C. illustrate the “ruggedness”of this novel variant dsRNA (i.e., Rugged dsRNA) and suggest that itwill be more bioavailable than most of the dsRNA molecules in aformulation of Ampligen®.

1. Bioactivity of Rugged dsRNA Shows Two-Fold Greater Binding Affinityas Compared to Unselected dsRNARugged dsRNA binding sites become unsaturated at a ratio of 0.50:1 (TLR3Rugged dsRNA) or higher. But binding sites for Ampligen® poly(I):poly(C₁₂U) become unsaturated at a ratio of 0.20:1 (TLR3:unselecteddsRNA) or higher.2. Stability of Rugged dsRNA is Four-Fold Greater than Unselected dsRNAAmpligen® poly(I): poly(C₁₂U) is stable (i.e., S_(w.20)>10.0) for lessthan 90 days when subjected to hydrolysis under thermal stress of 40° C.By contrast, Rugged dsRNA is stable for greater than 360 days under thesame conditions. Rugged dsRNA also has an increased resistance toribonuclease digestion.3. Stability and Bioactivity Data Show that Rugged dsRNA is MoreBioavailable than Unselected dsRNAFrom these stability and bioactivity considerations. Rugged dsRNA ismore bioavailable for the relevant signaling receptor that conveys thetherapeutic benefit. The Rugged dsRNA has the additional benefit ofmaintaining long-term stability at ambient temperatures, which hasimportant clinical implications for treating populations in regions ofthe world without adequate refrigeration capabilities.

Bioactivity Background

Toll-like receptors (TLR) are signalling molecules recognizingpathogen-associated molecular patterns (PAMP) and activating innateimmune defense mechanisms. TLR3 recognizes dsRNA, the genomic structureof some viruses, and also an intermediate generated during viral RNAreplication. dsRNA is also produced intracellularly by stem-loop formingor with siRNA-aligned mRNAs. Ampligen® is comprised of dsRNA moleculesthat act through TLR3 binding and downstream signaling events. Whilepoly(I):poly(C) signaling has alternate routes, the poly(I):poly(C₁₂U)pathway acts exclusively through TLR3 binding as Ampligen® treatmentprotects TLR3^(+/−) but not TLR3^(−/+) mice from Punta Toro virusinfection. TLR3^(−/−) cells do not produce IFN upon poly(I):poly(C₁₂U)treatment while IFN is induced by poly (I):poly(C) in TLR3 knockoutcells.

The TLR3 molecule ectodomain (ECD) conformation and its relation tobinding of dsRNA is well characterized, including the prospectivebinding site. Amino acids H539 and N541 are involved in the interactionwith the double helix. Mutational analysis of these amino acids at thebinding site further strengthens the argument.

The effect of length and structure of dsRNA on TLR3 binding and IFNinduction is know. Inosine₃₀ (I₃₀):poly(C) or poly(I):Cytosine₃₀ (C₃₀)induced interferon (IFN), but shorter dsRNA stretches do not induce IFN.Compared to them, however, IFN induction by poly(I):poly(C) was alwayssuperior. I₂₀:C₂₀, I₃₀:C₃₀ and I₄₀:C₄₀ were ineffective IFN inducers.Therefore, characterizing Ampligen® by its TLR3 binding capacity is abiomarker to predict its biological activity.

Bioactivity Method

A range of ratios of TLR3-ECD to unselected Ampligen® or Rugged dsRNAare reacted by the method of Leonard (2008). The components areseparated by the size-exclusion chromatographic method described below.From the peak quantities of free TLR3-ECD and the ligand-receptorcomplex, the ratio of TLR3-ECD that is required for saturation of eitherAmpiligen® or Rugged dsRNA is determined. This threshold TLR3-ECD/dsRNAratio provides a direct indication of the strength of theligand-receptor binding and, therefore, of bioactivity.

The following method is an adaptation of the experimental proceduresused to characterize TLR3 ligand binding at a molecular level. SinceTLR3-ECD (1.12×10² Kda) and poly(I):poly(C₁₂U) (0.2-2×10³ Kda) havedifferent elution patterns, they can be separated from each other bysize-exclusion chromatography (SEC). According to results obtained frompoly(I):poly(C) using a SUPERDEX 200 PC 3.2/30 column and collecting 80μl fractions, most of the poly(I):poly(C) appears in fractions 3-5 whileTLR3-ECD is eluted in fractions 9-12 (Bell 2005).

The binding of TLR3-ECD to poly(I):poly(C) or poly(I):poly(C₁₂U) createsa complex that is larger in size than either of the initial components.The later eluting free TLR3-ECD is separated from the complex.Optimization of the separation identified that the SUPEROSE 200 PCcolumn afforded superior binding by reducing tailing, due to absence ofnonspecific interactions with dsRNA.

FIG. 18 shows the resulting chromatograms obtained from one reactedmixture of TLR3-ECD/poly(I):poly(C₁₂U) compared to component injectionsat TLR3-ECD and poly(I):poly(C₁₂U) alone, respectively.

Characterization of Peaks. Identification and quantitation of TLR3-ECDin size-exclusion chromatography fractions is possible in an ELISAformat. The commercially-available TLR3-ECD is a His tag-containingrecombinant protein. A capture anti-His tag antibody immobilizesTLR3-ECD in a microplate well. A second, biotinylated primary antibodyquantitatively binds to the immobilized TLR3-ECD. This secondaryantibody is selected to have an epitope distal from the dsRNA bindingsite on the TLR3-ECD molecule and also from the epitope recognized bythe capture antibody. HRP-conjugated streptavidin recognizes thebiotinylated second primary antibody. The appropriate substratemetabolized by HRP produces a soluble color suitable for quantitativemeasurement of TLR3-ECD.

Ampligen® concentration in the size-exclusion chromatography fractionsis measured by fluorescence using standard dilutions and chromatographyfractions in a quantitative riboGreen test. This assay permits testingof Ampligen® out-of-the-bottle (i.e., not selected for Rugged dsRNA)without further processing, preparation, or extraction, therebymaintaining its condition as a pharmaceutical.

Bioactivity Results. Results in Table 6 show the percentage of freeTLR3-ECD that remains in a series of reactions using different ratios ofTLR3-ECD to dsRNA. These studies were conducted with either unimprovedAmpligen® as well as Rugged dsRNA.

Binding of TLR3-ECD to Rugged dsRNA is more effective than binding ofTLR3-ECD to unimproved Ampligen®. An approximately 2-fold greater ratioof TLR3-ECD is required to “unsaturate” Rugged dsRNA (−0.50:1) ascompared to Ampligen® (0.25:1). Also, the binding profile at variousratios shows a much sharper endpoint for saturation for the case ofRugged dsRNA which may reflect greater structural uniformity for thismore compact dsRNA Table 6. Bioactivity Measurements of UnimprovedAmpligen® vs. Rugged dsRNA.

Unimproved/Old Ampligen ®, Molar Lot # 0701HE New Rugged dsRNA Ratio ofdsRNA/TLR3 dsRNA/TLR3 TLR3 to Complex Free TLR3 Complex Free TLR3 dsRNAArea % Area % Area % Area % 0.20:1 99.0 0.978 99.4 0.577 0.25:1 78.421.6 99.1 0.880 0.33:1 20.9 79.1 92.9 7.086 0.50:1 58.9 41.1 60.3 39.7230.67:1 15.4 84.6 11.3 88.660

The TLR3 binding of Rugged dsRNA is 100% superior inin receptor bindingthan the unimproved/old Ampligen® preparation. As shown in Table 6, FreeTLR3 (area >10%) appears at a TLR3:dsRNA ratio of 0.25:1 for unimprovedAmpligen® as compared to a 0.50:1 for Rugged dsRNA

Stability of Rugged dsRNA. Stability of poly(I):poly(C₁₂U) was measuredat an accelerated temperature condition of 40° C. as compared to thelong-term storage temperature of from 2° C. to 8° C. As shown in FIG.19, the size of poly(I): poly(C₁₂U) decays at this temperature asmeasured by analytical ultracentrifugation (S_(20.w)). Decrease in sizeis due to unfolding of the double helix (loss of hydrogen bonds) andconcurrent hydrolysis of the phosphodiester bonds. For bioactivityunimproved Ampligen® (poly(I):poly(C₁₂U) requires a sedimentationcoefficient from about 10.0 to about 15.0 S(_(20.w)), whereas the sizeof poly(I):poly(C₁₂U) at more than 180 days is about 8.0 S(_(20.w)) andindicates a loss of bioactivity.

FIG. 20 shows the results of a second stability indicating parameter,the reversed phase HPLC assay, previously described, that separatespoly(I): poly(C₁₂U) into its individual strands (7 minute and 10 minutepeaks). It is clearly evident that hydrolysis begins with the poly(I)strand (10 minute peak) followed by the poly(C₁₂U) strand (7 minutepeak). HPLC results show that loss of size does not begin untilcommencement of the hydrolysis of the second strand poly(C₁₂U); the RNAmolecule retains double-stranded structure when only one of the strandsundergoes hydrolysis. This loss of size at about 90 days occurs with thehydrolysis of both poly(I) and poly(C₁₂) strands.

Importantly, the Rugged dsRNA (5 min) peak is entirely unaffected bythermal stress. In fact, it increases in relation to the poly(I) andpoly(C₁₂U) strands. This conclusively shows that Rugged dsRNA is notonly “rugged” but can form spontaneously from smaller strands ofdegraded poly(I):poly(C₁₂U).

Structure of Novel Improved Rugged dsRNA Compared to Old UnimprovedAmpligen® MixtureTransmission Electron Microscopy (TEM) was used to compare the structureof the new improved Rugged dsRNA vs. the old unimproved Ampligen®mixture of dsRNA molecules. As shown in FIGS. 26, 27, and 28 theunimproved Ampligen® mixture contains molecules with a high degree ofbranching compared to the new improved Rugged dsRNA (FIG. 29). In factthe majority of molecules in the Ampligen® mixture are branching, whilethe majority of dsRNA molecules in the improved Rugged dsRNA areunbranched. Also, the unimproved Ampligen® mixture contains 4-5 timesmore molecules with 3 or more branches than the new Reproved RuggeddsRNA. Therefore, not only does Rugged dsRNA contain a higher percentageof non-branched molecules. the small percentage of branched moleculespresent contain primarily a single branch as compared to the unimprovedAmpligen® mixture which contains many more highly branched variantmolecules with >3 branched strands. The binding affinity of dsRNA toTLR3 is a function of the length of the linear non-branched dsRNA. Thisexplains the increased bioactivity of Rugged dsRNA compared to the oldunimproved Ampligen® mixture.

Patents, patent applications, cooks, and other publications andinformation sources cited herein are incorporated by reference in theirentirety.

In stating a numerical range, it should be understood that all valueswithin the range are also described (e.g., one to ten also includesevery integer value between one and ten as well as all intermediateranges such as two to ten, one to five, and three to eight). The term“about” may refer to the statistical uncertainty associated with ameasurement or the variability in a numerical quantity which a personskilled in the art would understand does not affect operation of theinvention or its patentability.

All modifications and substitutions, that come within the meaning of theclaims and the range of their legal equivalents are to be embracedwithin their scope. A claim which recites “comprising” allows theinclusion of other elements to be within the scope of the claim; theinvention is also described by such claims reciting the transitionalphrases “consisting essentially of” (i.e., allowing the inclusion ofother elements to be within the scope of the claim if they do notmaterially affect operation of the invention) or “consisting of” (i.e.,allowing only the elements listed in the claim other than impurities orinconsequential activities which are ordinarily associated with theinvention) instead of the “comprising” term. Any of these threetransitions can be used to claim the invention.

It should be understood that an element described in this specificationshould not be construed as a limitation of the claimed invention unlessit is explicitly recited in the claims. Thus, the granted claims are thebasis for determining the scope of legal protection instead of alimitation from the specification which is read into the claims. Incontradistinction, the prior art is explicitly excluded from theinvention to the extent of specific embodiments that would anticipatethe claimed invention or destroy novelty.

Moreover, no particular relationship between or among limitations of aclaim is intended unless such relationship is explicitly recited in theclaim (e.g., the arrange-ment of components in a product claim or orderof steps in a method claim is not a limitation of the claim unlessexplicitly stated to be so). All possible combinations and permutationsof individual elements disclosed herein are considered to be aspects ofthe invention. Similarly, generalizations of the invention's descriptionare considered to be part of the invention.

From the foregoing, it would be apparent to a person of skill in thisart that the invention can be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments should be considered only as illustrative, not restrictive,because the scope of the legal protection provided for the inventionwill be indicated by the appended bases rather than by thisspecification.

REFERENCES

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1. An isolated double-stranded ribonucleic acid (dsRNA) which isresistant to denaturation under conditions that are able to separatehybridized poly(riboinosinic acid) and poly(ribocytosinic acid) strands,wherein said isolated dsRNA has at least one strand of a length of fromabout 40 bases to 375 bases.
 2. The isolated dsRNA of claim 1 whichcontains only partially hybridized strands.
 3. The isolated dsRNA ofclaim 1, wherein only a single strand of said isolated dsRNA comprisesone or more uracil or guanine bases that are not base paired to anopposite strand. 4.-8. (canceled)
 9. The isolated dsRNA of claim 1,wherein said isolated dsRNA is comprised ofribo(I_(n)).ribo(C₃₀₋₃₅U)_(n), in which ribo is a ribonucleotide and nis an integer from (40 to 500). 10.-11. (canceled)
 12. A compositioncomprising one or more different isolated dsRNAs as defined in claim 1and a carrier.
 13. An isolated double-stranded ribonucleic acid (dsRNA)which is resistant to denaturation under conditions that are able toseparate hybridized poly(riboinosinic acid) and poly(ribocytidylic acid)strands, wherein the isolated dsRNA: has an HPLC chromatogramsubstantially the same as the 5 minute peak of FIG. 1; is stable toexposure to thermal stress at 40° C; and has an increased bioactivity asevidenced by binding to receptor TLR3-ECD as compared to unimprovedpoly(I):poly(C₁₂U).
 14. An isolated double-stranded ribonucleic acid(dsRNA) which is resistant to enzymatic degradation under conditionsthat are able to degrade poly(riboinosinic acid) and poly(ribocytidylicacid) strands, wherein the isolated dsRNA: has an HPLC chromatogramsubstantially the same as the 5 minute peak of FIG. 1; has increasedstability to exposure to pancreatic ribonuclease A; and has an increasedbioactivity as evidenced by binding to receptor TLR3-ECD as compared tounselected poly(I):poly(C₁₂U).
 15. A pharmaceutical formulationcomprising dsRNA wherein at least 10 weight percent of said dsRNA is theisolated dsRNA according to claim
 1. 16.-20. (canceled)
 21. A method oftreating a subject with an immunological dysfunction, or a method oftreating or preventing tumor formation or viral infection in a subject,or a method of inducing an immune enhancing effect in a subject, saidmethod comprising administration to the subject of the isolated dsRNAdefined in claim 1 in a therapeutic amount. 22.-25. (canceled)
 26. Themethod according to claim 21, wherein the therapeutic amount of saidisolated dsRNA is infused intravenously, is injected intradermally,subcutaneously, or intramuscularly; inhaled intranasally orintratracheally; or is applied transdermally, transmucosally,intranasally, intratracheally, oropharyngeally, or sublingually. 27.-28.(canceled)
 29. An isolated double-stranded ribonucleic acid (dsRNA)which is resistant to denaturation under conditions that are able toseparate hybridized poly(riboinosinic acid) and poly(ribocytosinic acid)strands, wherein said dsRNA has a molecular weight from about 24 kda toabout 225 kda.
 30. The isolated dsRNA of claim 29 which contains onlypartially hybridized strands.
 31. The isolated dsRNA of claim 29,wherein only a single strand of said isolated dsRNA comprises one ormore uracil or guanine bases that are not base paired to an oppositestrand. 32.-36. (canceled)
 37. The isolated dsRNA of claim 29, whereinsaid isolated dsRNA is comprised of ribo(I_(n)).ribo(C₃₀₋₃₅U)_(n), inwhich ribo is a ribonucleotide and n is an integer from (40 to 500).38.-39. (canceled)
 40. A composition comprising one or more differentisolated dsRNAs as defined in claim 29 and a carrier.
 41. Apharmaceutical formulation comprising dsRNA wherein at least 10 weightpercent of said dsRNA is the isolated dsRNA according to claim 29.42.-45. (canceled)
 46. A method of treating a subject with animmunological dysfunction, or a method of treating or preventing tumorformation or viral infection in a subject, or a method of inducing animmune enhancing effect in a subject, said method comprisingadministration to the subject of the isolated dsRNA defined in claim 29in a therapeutic amount. 47.-50. (canceled)
 51. The method according toclaim 46, wherein the therapeutic amount said isolated dsRNA or isinfused intravenously, is injected intradermally, subcutaneously, orintramuscularly; inhaled intranasally or intratracheally; or appliedtransdermally, transmucosally, intranasally, intratracheally,oropharyngeally, or is sublingually. 52.-53. (canceled)
 54. An isolateddouble-stranded ribonucleic acid (dsRNA) which is resistant todenaturation under conditions that are able to separate hybridizedpoly(riboinosinic acid) and poly(ribocytosinic acid) strands, whereinsaid isolated dsRNA is comprised of ribo(I_(n)).ribo(C₃₀₋₃₅U)_(n), inwhich ribo is a ribonucleotide and n is an integer from (40 to 500). 55.The isolated dsRNA of claim 54 which contains only partially hybridizedstrands.
 56. The isolated dsRNA of claim 54, wherein only a singlestrand of said isolated dsRNA comprises one or more uracil bases thatare not base paired to an opposite strand. 57.-60. (canceled)
 61. Theisolated dsRNA of claim 54, wherein said isolated dsRNA is comprised ofribo(I_(n)).ribo(C₃₀U)_(n), in which ribo is a ribonucleotide and n isan integer from 40 to
 500. 62.-64. (canceled)
 65. A compositioncomprising one or more different isolated dsRNAs as defined in claim 54and a carrier. 66.-71. (canceled)
 72. A method of treating a subjectwith an immunological dysfunction, or a method of treating or preventingtumor formation or viral infection in a subject, or a method of inducingan immune enhancing effect in a subject, said method comprisingadministration to the subject of the isolated dsRNA defined in claim 54in a therapeutic amount. 73.-76. (canceled)
 77. The method according toclaim 72, wherein the therapeutic amount of said isolated dsRNA isinfused intravenously, is injected intradermally, subcutaneously, orintramuscularly; inhaled intranasally or intratracheally; or is appliedtransdermally, transmucosally, intranasally, intratracheally,oropharyngeally, or sublingually. 78.-79. (canceled)